Microfluidic device for storage and well-defined arrangement of droplets

ABSTRACT

The present invention relates to systems and methods for the arrangement of droplets in pre-determined locations. Many applications require the collection of time-resolved data. Examples include the screening of cells based on their growth characteristics or the observation of enzymatic reactions. The present invention provides a tool and related techniques which addresses this need, and which can be used in many other situations. The invention provides, in one aspect, a tool that allows for stable storage and indexing of individual droplets. The invention can interface not only with microfluidic/microscale equipment, but with macroscopic equipment to allow for the easy injection of liquids and extraction of sample droplets, etc.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/496,750, filed Apr. 25, 2017, entitled “Microfluidic Device ForStorage and Well-Defined Arrangement of Droplets” which is a divisionalof U.S. patent application Ser. No. 12/990,102, with a § 371 date ofMar. 16, 2011, entitled “Microfluidic Device For Storage andWell-Defined Arrangement of Droplets,” which is a national stage filingof Int. Patent Application Serial No. PCT/US2009/002644, filed Apr. 28,2009, which claims the benefit of U.S. Provisional Patent ApplicationSer. No. 61/048,304, filed Apr. 28, 2008, entitled “Microfluidic Devicefor Storage and Well-Defined Arrangement of Droplets,” by Weitz, et al.,each of which is incorporated herein by reference in its entirety.

FIELD OF INVENTION

The present invention relates generally to systems and methods for thearrangement of droplets in pre-determined locations.

BACKGROUND

Droplet-based microfluidics is a powerful emerging set of techniqueswith a great deal of application in chemistry and biology. Droplets canbe merged with other droplets, split into multiple droplets, sorted,and/or used to house reactions for example. Droplet-based microfluidictechniques allow for the production and handling of a very high volumeof droplets per unit of time. Hence, droplet-based microfluidic devicesare an ideal tool for high-throughput applications.

A number of patent applications describe formation and/or use ofdroplets for these and other procedures. A representative list includes:U.S. patent application Ser. No. 11/246,911, filed Oct. 7, 2005,entitled “Formation and Control of Fluidic Species,” published as U.S.Patent Application Publication No. 2006/0163385 on Jul. 27, 2006; U.S.patent application Ser. No. 11/024,228, filed Dec. 28, 2004, entitled“Method and Apparatus for Fluid Dispersion,” published as U.S. PatentApplication Publication No. 2005/0172476 on Aug. 11, 2005; U.S. patentapplication Ser. No. 11/360,845, filed Feb. 23, 2006, entitled“Electronic Control of Fluidic Species,” published as U.S. PatentApplication Publication No. 2007/0003442 on Jan. 4, 2007; InternationalPatent Application No. PCT/US2006/007772, filed Mar. 3, 2006, entitled“Method and Apparatus for Forming Multiple Emulsions,” published as WO2006/096571 on Sep. 14, 2006; U.S. patent application Ser. No.11/221,585, filed Sep. 8, 2005, entitled “Microfluidic Manipulation ofFluids and Reactions,” published as U.S. Patent Application PublicationNo. 2007/0052781 on Mar. 8, 2007; U.S. patent application Ser. No.11/698,298, filed Jan. 24, 2007, entitled “Fluidic Droplet Coalescence,”published as U.S. Patent Application Publication No. 2007/0195127 onAug. 23, 2007; and U.S. Provisional Patent Application Ser. No.60/920,574, filed Mar. 28, 2007, entitled “Multiple Emulsions andTechniques for Formation,” each incorporated herein by reference.

Although many advances associated with formation and use of dropletshave been achieved, there is a need for improved techniques and tools.

SUMMARY OF THE INVENTION

The present invention relates generally to systems and methods for thearrangement of droplets in pre-determined locations. The subject matterof the present invention involves, in some cases, interrelated products,alternative solutions to a particular problem, and/or a plurality ofdifferent uses of one or more systems and/or articles.

In one aspect, the invention is directed to a method or arrangingdroplets at predetermined positions. In a first set of embodiments, themethod comprises urging a first droplet through a first constriction ina channel into a first pot. In some cases, the method further comprisesapplying a pressure differential along the channel such that the firstdroplet is urged through a second constriction into a second pot that isdownstream of the first pot. In some instances, the method furthercomprises applying a pressure differential along the channel such that asecond droplet is urged through the first constriction into the firstpot wherein upon removal of the applied pressure differential, the firstdroplet is contained within the second pot and the second droplet iscontained within the first pot simultaneously.

In another set of embodiments, the method comprises applying a pressuredifferential along a channel that does not branch such that a firstdroplet is urged through the channel into a first pot. In some cases,the method further comprises applying a pressure differential such thatthe first droplet is passed through the channel that does not branchinto a second pot that is downstream of the first pot. In additionalcases, the method further comprises applying a pressure differentialsuch that a second droplet is passed through the channel that does notbranch into the first pot wherein the first droplet is contained withinthe second pot and the second droplet is contained within the first potsimultaneously.

In another set of embodiments, the method comprises applying a pressuredifferential in one direction such that a first droplet is urged througha first constriction into a first pot and subsequently urged through asecond constriction from the first pot into a second pot. In some cases,the method further comprises applying a pressure differential in onedirection such that a second droplet is passed through the channel intothe first pot wherein the first droplet is contained within the secondpot and the second droplet is contained within the first potsimultaneously.

In another set of embodiments, the method comprises applying anessentially constant pressure gradient along a channel such that a firstdroplet is urged through a first constriction into a first pot and thenurged through a second constriction from the first pot into a secondpot. In some cases, the method further comprises continuing to apply theessentially constant pressure gradient such that a second droplet isurged through the first constriction into the first pot wherein thefirst droplet is contained within the second pot and the second dropletis contained within the first pot simultaneously.

In another set of embodiments, the method comprises applying a pressuredifferential along a channel comprising a series of pots each containingat least one droplet, said pots connected via a series of constrictionsarranged such that each pot is in direct fluid communication with nomore than two other pots and each pot is connected to exactly twoconstrictions, and ejecting the droplets from the exit of the channel inthe order in which they were placed in the pots.

In another set of embodiments, the method comprises urging a first cellthrough a first constriction in a channel into a first pot. In somecases, the method further comprises applying a pressure differentialalong the channel such that the first cell is urged through a secondconstriction into a second pot that is downstream of the first pot. Insome instances, the method further comprises applying a pressuredifferential along the channel such that a second cell is urged throughthe first constriction into the first pot wherein, upon removal of theapplied pressure differential, the first cell is contained within thesecond pot and the second cell is contained within the first potsimultaneously.

In another aspect, the invention is directed to a method of fabricatinga device for arranging droplets. In one set of embodiments, the methodcomprises using a single lithography step to construct a channelcomprising a series of pots connected via a series of constrictionsarranged such that each pot is in direct fluid communication with nomore than two other pots and each pot is connected to exactly twoconstrictions.

In some embodiments, the method comprises urging a plurality of dropletstoward a channel comprising a plurality of pots, wherein at least about1% of the droplets are immobilized within pots. The method may furthercomprise, in some embodiments, loading droplets into pots at a rate ofat least about 10 droplets per second.

In another aspect, the invention is directed to a device for arrangingdroplets. In one set of embodiments, the device comprises a channelcomprising a series of pots connected via a series of constrictionsarranged such that each pot is in direct fluid communication with nomore than two other pots and each pot is connected to exactly twoconstrictions.

Other advantages and novel features of the present invention will becomeapparent from the following detailed description of various non-limitingembodiments of the invention when considered in conjunction with theaccompanying figures. In cases where the present specification and adocument incorporated by reference include conflicting and/orinconsistent disclosure, the present specification shall control. If twoor more documents incorporated by reference include conflicting and/orinconsistent disclosure with respect to each other, then the documenthaving the later effective date shall control.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting embodiments of the present invention will be described byway of example with reference to the accompanying figures, which areschematic and are not intended to be drawn to scale. In the figures,each identical or nearly identical component illustrated is typicallyrepresented by a single numeral. For purposes of clarity, not everycomponent is labeled in every figure, nor is every component of eachembodiment of the invention shown where illustration is not necessary toallow those of ordinary skill in the art to understand the invention. Inthe figures:

FIG. 1 is a schematic illustrating of an article including an array ofpots according to one embodiment of the invention.

FIGS. 2A-D are schematics illustrating the placement of droplets in potsaccording to one embodiment of the invention.

FIG. 3 is an optical micrograph of an article according to oneembodiment of the invention.

FIG. 4 is an optical micrograph of an article according to oneembodiment of the invention.

FIGS. 5A-5B include (a) an optical micrograph of an article according toone embodiment of the invention during an example experiment and (b) aplot of generation time versus the number of initially encapsulatedcells during an example experiment.

FIG. 6 includes an illustration of an article including an array of potsaccording to one embodiment of the invention.

FIGS. 7A-7B include (a) an optical micrograph of an article according toone embodiment of the invention during an example experiment and (b) aplot of the number of cells as a function of time during an exampleexperiment.

FIGS. 8A-8C include (a) an optical micrograph and color map gradientfrom an example experiment, (b) a plot of relative fluorescence as afunction of time in an example experiment, and (c) a plot of the numberof droplets with various rates of fluorescein production in an exampleexperiment.

DETAILED DESCRIPTION

The present invention relates to systems and methods for the arrangementof droplets in pre-determined locations. Many applications require thecollection of time-resolved data. Examples include the screening ofcells based on their growth characteristics or the observation ofenzymatic reactions. The present invention provides tools and relatedtechniques which address this need, and which can be used in many othersituations. The invention provides, in one aspect, a tool that allowsfor stable storage and indexing of individual droplets. The inventioncan interface not only with microfluidic/microscale equipment, but withmacroscopic equipment to allow for the easy injection of liquids andextraction of sample droplets, etc.

In one aspect, an inventive device has been fabricated that includes oneor more “pots” into which individual droplets can be transported andstored. Another aspect includes methods for arranging droplets in pots.In one embodiment, a droplet is urged through a constriction in astorage channel into a pot. Once in the pot, the droplet may remainstably positioned, or it may be urged from the pot through a secondconstriction and/or through further constrictions into and/or throughvarious pots which can identical or similar to, or different from, theoriginal pot.

The systems and methods of the invention can be used in a variety ofapplications that can benefit from the ability to immobilize droplets.For example, nanoliter-scale pots may allow for the observation of many,e.g., thousands, of chemical reactions on a single microfabricated chipwhere, for example, individual reactions are contained within individualpots (within droplets within those pots) associated with a chip. In someinstances, cells can be contained within a droplet, and the cells can bestored and/or delivered and, within pots, reactions involving cells canbe carried out and/or observed and/or other cellular interactions (e.g.,production of products from cells) can be carried out and/or observed.In some embodiments, the cells themselves may constitute droplets, andthe cells may be stored within and/or delivered to pots fluid. Otherspecies that can be stored and/or delivered include, for example,biochemical species such as nucleic acids such as siRNA, RNAi and DNA,proteins, peptides, or enzymes. Additional species that can beincorporated within a droplet of the invention include, but are notlimited to, nanoparticles, quantum dots, indicators, dyes, fluorescentspecies, chemicals, or the like. Droplets can also serve as reactionvessels in certain cases, such as for controlling chemical reactions, orfor in vitro transcription and translation, e.g., for directed evolutiontechnology.

Embodiments are described generally herein with reference to a set offluidic droplets, carried in a second fluid, and urged throughconstrictions into pots in which they can reside in the absence ofanother urging force or below a threshold force. In all embodimentsherein it is to be understood that droplets of a first fluid, carried ina second, continuous fluid can be replaced by other conformable objectssuch as cells (described in greater detail herein) which can, but neednot be, carried in a carrier liquid. For example, cells or otherconformable objects can be arranged in pots of the invention surroundedby another fluidic substance, a gas, or the like. Examples ofconformable substances include cells, gels, deformable polymers, and ofthe like.

FIG. 1 is a schematic of a device 10 according to one embodiment of theinvention. The illustrative embodiment includes a storage channel 12,e.g., an essentially straight storage channel, comprising a series ofpots (20, 22, and 24) and constrictions (40, 42, 44, and 46). As usedherein, the term “pot” refers to a portion of a storage channel with amaximum cross-sectional area that is larger than the maximumcross-sectional area of a portion or portions of the storage channeldirectly adjacent to the pot. In some embodiments, pots are spherical inshape with the exception, of course, of portions of the essentiallyspherical pot defining inlets and/or outlets (e.g., constrictions asshown in FIG. 1). In some embodiments, pots may be other shapesincluding cylinders, cubes, and cuboids, among others. Devices describedherein may include any number of pots. In some cases, a device comprisesa single pot. In other instances, a device may comprise at least 100, atleast 1000, or at least 10,000 pots. In addition, the pots describedherein may be of any volume. In some cases, the volumes of one or morepots are less than about 100 femtoliters, less than about 500femtoliters, less than about 5 picoliters, less than about 100picoliters, less than about 500 picoliters, less than about 1 nanoliter,or less than about 10 nanoliters.

Pots may be used to store droplets (30, 32, 34). The term “droplet,” asused herein, refers to an isolated portion of a first fluid that issurrounded by a second fluid, where the first and second fluids areimmiscible on the time scale of use of the device of the invention. Asused herein, the term “fluid” generally refers to a substance that tendsto flow and to conform to the outline of its container. Typically,fluids are materials that are unable to withstand a static shear stress,and when a shear stress is applied, the fluid experiences a continuingand permanent distortion. The fluid may have any suitable viscosity thatpermits at least some flow of the fluid. Non-limiting examples of fluidsinclude liquids and gases, but may also include free-flowing solidparticles (e.g., cells, vesicles, etc.), viscoelastic fluids, and thelike. Making and using such droplets, including use in a variety ofchemical, biological or biochemical settings, are described in variousdocuments including U.S. patent application Ser. No. 11/643,151, filedDec. 20, 2006, entitled “Compartmentalised combinatorial chemistry bymicrofluidic control,” published as U.S. Patent Application PublicationNo. 2007/0184489 on Aug. 9, 2007 and in International Patent ApplicationNo. PCT/US2006/007772, filed Mar. 3, 2006, entitled “Method andApparatus for Forming Multiple Emulsions,” published as WO 2006/096571on Sep. 14, 2006, incorporated herein by reference. In some preferredembodiments, the droplet(s) are spherical. In some embodiments, thedroplet(s) are not necessarily spherical, but may assume other shapes aswell, for example, depending on the external environment (e.g., a shapeof a pot within which a droplet is contained; typically, when a dropletis passed through a constriction defining an inlet to a pot, it assumesa shape different than that which it assumes within the pot). In oneembodiment, a droplet will have a maximum cross-sectional dimension thatis larger than the smallest dimension of a constriction perpendicular tofluid flow in which the droplet is located. When a population ofdroplets is used, the droplet(s) in such an arrangement will have anaverage cross-sectional dimension that is larger than the smallestdimension of a constriction perpendicular to fluid flow in which thedroplet is located. In this context, the cross-sectional dimension of adroplet is such a dimension when the droplet is not physicallyconstrained by, for example, a constriction or inlet to a pot, but onein which the droplet's shape is controlled by the free energy of thedroplet and its surrounding environment. As mentioned above, thedroplet(s) may contain additional entities or species which may be anysubstance that can be contained in any portion of a droplet and can bedifferentiated from the droplet fluid.

In cases where multiple droplets are present, the droplets may each besubstantially the same shape and/or size (“monodisperse”). The shapeand/or size of the droplets can be determined, for example, by measuringthe average diameter or other characteristic dimension of the droplets.The average diameter of a droplet (and/or of a plurality or series ofdroplets) may be, for example, less than about 1 mm, less than about 500micrometers, less than about 200 micrometers, less than about 100micrometers, less than about 75 micrometers, less than about 50micrometers, less than about 25 micrometers, less than about 10micrometers, or less than about 5 micrometers in some cases. The averagediameter may also be at least about 1 micrometer, at least about 2micrometers, at least about 3 micrometers, at least about 5 micrometers,at least about 10 micrometers, at least about 15 micrometers, at leastabout 20 micrometers, or at least about 100 micrometers in certaincases.

As noted, in the illustrative embodiment are a series of constrictions(40, 42, 44, and 46 in FIG. 1). As used herein, the term “constriction”refers to a portion of a storage channel with a cross-sectional areathat is smaller than those of the portions of the storage channeldirectly adjacent to the constriction, e.g., pots. In order to promoteconfining the droplets to pots, the minimum cross-sectional diameters ofthe constrictions may be smaller than the maximum cross-sectionaldiameters of the adjacent pots. In some embodiments, the minimumcross-sectional diameters of the constrictions are less than 50% of themaximum cross-sectional diameters of the adjacent pots. In someembodiments, the minimum cross-sectional diameters of the constrictionsare less than 25%, less than 10%, or less than 5% of the maximumcross-sectional diameters of the adjacent pots.

While description of the relationship between pots, constrictions, anddroplets is provided, it should be understood that in one embodiment allpots of a single storage channel, or all pots of an entire device, areof the same size, with all constrictions of a single storage channel orof an entire device being the same size, and all droplets areessentially the same size, but in other arrangements different potsizes, different constriction sizes and/or different droplets can beused. Those of ordinary skill in the art will understand that suchvariability can occur for a variety of purposes, and will understandthat wherever single sizes of any of the above are described multiplesizes can be selected. Additionally, any number of pots can be used in asingle storage channel, and any number of storage channels can be used,in series or parallel, in a particular device.

The devices described herein comprise, in one set of embodiments, one ormore storage channels. A “storage channel,” as used herein, means afeature on or in an article (substrate) that at least partially directsflow of a fluid and comprises one or more pots connected via a series ofat least two constrictions. The channel can have any cross-sectionalshape (circular, oval, triangular, irregular, square or rectangular, orthe like) and can be enclosed or unenclosed. In embodiments where it iscompletely enclosed, at least one portion of the channel can have across-section that is completely enclosed, or the entire channel may becompletely enclosed along its entire length with the exception of itsinlet(s) and/or outlet(s). A channel may also have an aspect ratio(length to average cross sectional dimension) of at least 2:1, moretypically at least 3:1, 5:1, 10:1, 50:1, 100:1 or more. An unenclosedchannel generally will include characteristics that facilitate controlover fluid transport, e.g., structural characteristics (an elongatedindentation) and/or physical or chemical characteristics (hydrophobicityvs. hydrophilicity) or other characteristics that can exert a force(e.g., a containing force) on a fluid. The fluid within the channel maypartially or completely fill the channel. In some cases where anunenclosed channel is used, the fluid may be held within the channel,for example, using surface tension (i.e., a concave or convex meniscus).

The channel may be of any size, for example, having a largest dimensionperpendicular to fluid flow of less than about 5 mm or 2 mm, or lessthan about 1 mm, or less than about 500 microns, less than about 200microns, less than about 100 microns, less than about 60 microns, lessthan about 50 microns, less than about 40 microns, less than about 30microns, less than about 25 microns, less than about 10 microns, lessthan about 3 microns, less than about 1 micron, less than about 300 nm,less than about 100 nm, less than about 30 nm, or less than about 10 nm.The dimensions of the channel may also be chosen, for example, to allowa certain volumetric or linear flowrate of fluid through the channel. Ofcourse, the number of channels and the shape of the channels can bevaried by any method known to those of ordinary skill in the art.

In some embodiments, including that shown in FIG. 1, the pots andconstrictions in the storage channel are arranged such that each pot isin direct fluid communication with no more than two other pots, and eachpot is connected to exactly two constrictions. In some embodiments, thestorage channels comprising pots and constrictions do not branch. Insome cases, the storage channels do not branch between the first andlast pots in the storage channel. The term “branch,” as used herein,means to divide or separate into two or more parts or subdivisions. Thestorage channels of the device may, in some instances, be substantiallystraight. In some embodiments, the storage channels may be serpentine,circular, or follow any other type of path.

A variety of materials and methods, according to certain aspects of theinvention, can be used to form systems described herein. In some cases,the various materials selected lend themselves to various methods. Forexample, various components of the invention can be formed from solidmaterials, in which the storage channels can be formed viamicromachining, film deposition processes such as spin coating andchemical vapor deposition, laser fabrication, photolithographictechniques, etching methods including wet chemical or plasma processes,and the like. See, for example, Scientific American, 248:44-55, 1983(Angell, et al). In one set of embodiments, a single lithography stepmay be used to construct the devices described herein. In oneembodiment, the devices and/or molds used to produce the devicesdescribed herein may be microfabricated using a single bulk etchingstep. For example, in one set of embodiments laser etching could be usedto make devices in a one-step process. As another example, in one set ofembodiments a master may be fabricated using standard photolithography.The structure on the master may be essentially the same heighteverywhere. The master may be molded in PDMS. In this case, since theresulting channels would have essentially the same height throughout thedevice, several layers of photoresist and/or several layers of PDMSwould not be needed. In one embodiment, at least a portion of thefluidic system is formed of silicon by etching features in a siliconchip. Technologies for precise and efficient fabrication of variousfluidic systems and devices of the invention from silicon are known. Inanother embodiment, various components of the systems and devices of theinvention can be formed of a polymer, for example, an elastomericpolymer such as polydimethylsiloxane (“PDMS”), polytetrafluoroethylene(“PTFE” or Teflon®), or the like.

Different components can be fabricated of different materials. Forexample, a base portion including a bottom wall and side walls can befabricated from an opaque material such as silicon, and a top portioncan be fabricated from a transparent or at least partially transparentmaterial, such as glass or a transparent and/or partially transparentpolymer (e.g., PDMS), for observation and/or control of the fluidicprocess. Components can be coated so as to expose a desired chemicalfunctionality to fluids that contact interior storage channel walls,where the base supporting material does not have a precise, desiredfunctionality. For example, components can be fabricated as illustrated,with interior storage channel walls coated with another material.Material used to fabricate various components of the systems and devicesof the invention, e.g., materials used to coat interior walls of fluidstorage channels, may desirably be selected from among those materialsthat will not adversely affect or be affected by fluid flowing throughthe fluidic system, e.g., material(s) that is chemically inert in thepresence of fluids to be used within the device.

In one embodiment, various components of the invention are fabricatedfrom polymeric and/or flexible and/or elastomeric materials, and can beconveniently formed of a hardenable fluid, facilitating fabrication viamolding (e.g. replica molding, injection molding, cast molding, etc.).The hardenable fluid can be essentially any fluid that can be induced tosolidify, or that spontaneously solidifies, into a solid capable ofcontaining and/or transporting fluids contemplated for use in and withthe fluidic network. In one embodiment, the hardenable fluid comprises apolymeric liquid or a liquid polymeric precursor (i.e. a “prepolymer”).Suitable polymeric liquids can include, for example, thermoplasticpolymers, thermoset polymers, or mixture of such polymers heated abovetheir melting point. As another example, a suitable polymeric liquid mayinclude a solution of one or more polymers in a suitable solvent, whichsolution forms a solid polymeric material upon removal of the solvent,for example, by evaporation. Such polymeric materials, which can besolidified from, for example, a melt state or by solvent evaporation,are well known to those of ordinary skill in the art. A variety ofpolymeric materials, many of which are elastomeric, are suitable, andare also suitable for forming molds or mold masters, for embodimentswhere one or both of the mold masters is composed of an elastomericmaterial. A non-limiting list of examples of such polymers includespolymers of the general classes of silicone polymers, epoxy polymers,and acrylate polymers. Epoxy polymers are characterized by the presenceof a three-membered cyclic ether group commonly referred to as an epoxygroup, 1,2-epoxide, or oxirane. For example, diglycidyl ethers ofbisphenol A can be used, in addition to compounds based on aromaticamine, triazine, and cycloaliphatic backbones. Another example includesthe well-known Novolac polymers. Non-limiting examples of siliconeelastomers suitable for use according to the invention include thoseformed from precursors including the chlorosilanes such asmethylchlorosilanes, ethylchlorosilanes, phenylchlorosilanes, etc.

Silicone polymers are preferred in one set of embodiments, for example,the silicone elastomer polydimethylsiloxane. Non-limiting examples ofPDMS polymers include those sold under the trademark Sylgard by DowChemical Co., Midland, Mich., and particularly Sylgard 182, Sylgard 184,and Sylgard 186. Silicone polymers including PDMS have severalbeneficial properties simplifying fabrication of the microfluidicstructures of the invention. For instance, such materials areinexpensive, readily available, and can be solidified from aprepolymeric liquid via curing with heat. For example, PDMSs aretypically curable by exposure of the prepolymeric liquid to temperaturesof about, for example, about 65° C. to about 75° C. for exposure timesof, for example, about an hour. Also, silicone polymers, such as PDMS,can be elastomeric, and thus may be useful for forming very smallfeatures with relatively high aspect ratios, necessary in certainembodiments of the invention. Flexible (e.g., elastomeric) molds ormasters can be advantageous in this regard.

One advantage of forming structures such as microfluidic structures ofthe invention from silicone polymers, such as PDMS, is the ability ofsuch polymers to be oxidized, for example by exposure to anoxygen-containing plasma such as an air plasma, so that the oxidizedstructures contain, at their surface, chemical groups capable ofcross-linking to other oxidized silicone polymer surfaces or to theoxidized surfaces of a variety of other polymeric and non-polymericmaterials. Thus, components can be fabricated and then oxidized andessentially irreversibly sealed to other silicone polymer surfaces, orto the surfaces of other substrates reactive with the oxidized siliconepolymer surfaces, without the need for separate adhesives or othersealing means. In most cases, sealing can be completed simply bycontacting an oxidized silicone surface to another surface without theneed to apply auxiliary pressure to form the seal. That is, thepre-oxidized silicone surface acts as a contact adhesive againstsuitable mating surfaces. Specifically, in addition to beingirreversibly sealable to itself, oxidized silicone such as oxidized PDMScan also be sealed irreversibly to a range of oxidized materials otherthan itself including, for example, glass, silicon, silicon oxide,quartz, silicon nitride, polyethylene, polystyrene, glassy carbon, andepoxy polymers, which have been oxidized in a similar fashion to thePDMS surface (for example, via exposure to an oxygen-containing plasma).Oxidation and sealing methods useful in the context of the presentinvention, as well as overall molding techniques, are described in theart, for example, in an article entitled “Rapid Prototyping ofMicrofluidic Systems and Polydimethylsiloxane,” Anal. Chem., 70:474-480,1998 (Duffy, et al.), incorporated herein by reference.

In some embodiments, certain microfluidic structures of the invention(or interior, fluid-contacting surfaces) may be formed from certainoxidized silicone polymers. Such surfaces may be more hydrophilic thanthe surface of an elastomeric polymer. Such hydrophilic storage channelsurfaces can thus be more easily filled and wetted with aqueoussolutions.

In one embodiment, a bottom wall of a microfluidic device of theinvention is formed of a material different from one or more side wallsor a top wall, or other components. For example, the interior surface ofa bottom wall can comprise the surface of a silicon wafer or microchip,or other substrate. Other components can, as described above, be sealedto such alternative substrates. Where it is desired to seal a componentcomprising a silicone polymer (e.g. PDMS) to a substrate (bottom wall)of different material, the substrate may be selected from the group ofmaterials to which oxidized silicone polymer is able to irreversiblyseal (e.g., glass, silicon, silicon oxide, quartz, silicon nitride,polyethylene, polystyrene, epoxy polymers, and glassy carbon surfaceswhich have been oxidized). Alternatively, other sealing techniques canbe used, as would be apparent to those of ordinary skill in the art,including, but not limited to, the use of separate adhesives, ‘bonding,solvent bonding, ultrasonic welding, etc.

In another aspect, the present invention relates generally to methodsfor the arrangement of droplets in pre-determined locations. FIGS. 2A-Dillustrate a method, according to one embodiment, of arranging dropletsin pots. In this embodiment, first droplet 30 is urged through firstconstriction 40 and into first pot 20. As seen in FIG. 2A, first droplet30 is deformed as it is urged through constriction 40. The deformationof droplet 30 as illustrated in FIG. 2A may be achieved by applying apressure gradient across the droplet. A “pressure gradient,” as definedherein, describes a situation in a fluid continuum in which a point ofrelatively high pressure and a point of relatively low pressure existsimultaneously and the points of relatively high and relatively lowpressure are on opposite sides of the droplet. Typically, a pressuregradient will exist between the inlet of a storage channel and theoutlet of the channel. Pressure gradients in the system may be appliedby those methods known in the art such as, for example, through the useof syringe pumps. In some cases, the pressure gradient across thechannel may be very accurately controlled using such methods. In theembodiments outlined in FIGS. 2A-D, a pressure gradient is applied suchthat the pressure decreases in the direction of arrow 50. In FIG. 2B,droplet 30 has entered pot 20 and relaxed to its free energy (in theembodiment illustrated, and essentially spherical) shape. If thepressure gradient is reduced below a critical value or completelyeliminated, droplet 30 may remain confined in pot 20. For example, thepressure gradient may be eliminated by simultaneously disconnecting allexternal tubing (e.g., by pulling it out of the device or cutting itoff), thereby exposing all channel openings to the same atmosphericpressure. Alternatively, droplet 30 may be urged through constriction42, as shown in FIG. 2C. In some embodiments, second droplet 32 may beurged through first constriction 40 as first droplet 30 issimultaneously urged through second constriction 42. Eventually, seconddroplet 32 will enter first pot 20, and first droplet 30 will entersecond pot 22. Both droplets will relax to their natural round shapeonce they have entered the pots as shown in FIG. 2D.

If the pressure gradient is reduced below a critical value or completelyeliminated, the droplets will remain confined in their pots. Firstdroplet 30 may, in some cases, be urged through third constriction 44into third pot 24, second droplet 32 may be urged through secondconstriction 42 into second pot 22, and third droplet 34 may be urgedthrough first constriction 40 into first pot 20. This process may berepeated for any number of pots and/or droplets. FIGS. 3-4 includeoptical micrographs of droplets arranged in an array of pots accordingto one embodiment.

In some embodiments, the pressure gradient used to urge droplets intopots remains substantially constant in direction, magnitude, or both. Asused herein, the phrase “constant pressure gradient” refers to pressuregradient does not change in magnitude or direction with time.

Based on the description herein, those of ordinary skill in the art canreadily position a variety of droplets in a variety of pots in a staticor dynamic arrangement. That is, using pressure gradients any number ofdroplets can be positioned in any number of specific pots and thenanalyzed or used for any of a variety of purposes. Similarly, dropletscan be move through storage channels in a controlled manner. Forexample, a pressure gradient can be selected to move droplets throughconstrictions into successive pots at a rate at which droplets willremain in post for a length of time sufficient to carry out a particularprocedure. That is, at a particular pressure gradient a droplet may passfrom one pot through a constriction into a downstream pot but thenremain in the downstream pot for a period of time, then be urged througha subsequent constriction into a subsequent pot but similarly remain inthe subsequent pot for a particular period of time. In this manner, thepressure gradient can be controlled such that droplets move frompot-to-pot but remain in pots long enough for an analysis, reaction, orother technique to be carried out.

In some embodiments, droplets may be loaded into pots at relatively highefficiencies, which is to say, a relatively large number of dropletsthat are urged toward a channel comprising pots are immobilized withinthe pots. A droplet may not be immobilized within a pot for a variety ofreasons. For example, the droplet may flow through a bypass channel (anexample of which is described in Example 1) rather than through achannel comprising pots. In some cases, a droplet could rupture or mergewith another droplet. As another example, a droplet may be leaked out ofthe device or the loading instrument (e.g., a pipette, syringe, etc.)over the time scale of the loading process. In some embodiments, aplurality of droplets are urged toward a channel comprising a pluralityof pots, and at least about 1%, at least about 10%, at least about 25%,at least about 50%, at least about 75%, at least about 90%, at leastabout 95%, or at least about 99% of the droplets are immobilized withinpots. In some embodiments, droplets are produced within a devicecomprising a storage channel. In such cases, urging the droplets towarda channel comprising a plurality of pots may comprise exerting apressure on the fluid containing the droplets such that the fluidcontaining the droplets is flowed into the channel comprising theplurality of pots. In some cases, urging droplets toward a channelcomprising a plurality of pots may comprise loading droplets into thedevice comprising the storage channel via a pipette, syringe, or otherexternal instrument. In such cases, a droplet is said to be urged towarda channel comprising a plurality of pots once the droplet exits thepipette, syringe, or other external container.

Droplets may be urged into pots at a relatively fast rate. For example,in some cases, droplets may be urged into pots at a rate of at leastabout 10, at least about 100, at least about 1000, or at least about10,000 droplets per second. Droplets may be urged into pots at highrates (e.g., any of the rates listed above) while maintaining any of theabove loading efficiencies, in some embodiments.

In another set of embodiments, a method for removing droplets from potsis described. In some cases, droplets can be removed one at a time, inthe order in which the droplets entered the storage channel. This may beachieved, for example, by applying a pressure gradient. The pressuregradient may be constant in direction, magnitude, or both. In somecases, a third fluid may be provided at the exit of the storage channel.Constant volumes of the third fluid may be provided after the exit of anumber of droplets, in some instances, in order to maintain constantspacing between droplets or sets of droplets. In some cases, constantvolumes of fluid may be provided after each droplet exits the storagechannel.

In some cases, the droplets may be removed from the pots and transportedto a secondary device for analysis. The method of removing the dropletsfrom pots may, in some embodiments, comprise applying a pressuregradient along the storage channel and urging the droplets through theexit one at a time. In other embodiments, a hole may be formed adjacentto the pot and the contents of the pot may be drained into a secondarydevice. Examples of secondary devices include, but are not limited to,microtiter plates (96-well, 140-well, 384-well, 1536-well, etc.), DNAmicroarrays (also known as a gene or genome chip, DNA chip, or genearray), protein arrays, chemostats, fluorescence activated cell sorters(FACS), flow cytometers, microreactors, and microfluidic devices, amongothers. As one example, holes may be formed adjacent to the pots of onedevice, and the contents of those pots may be transferred into the wellsof a microtiter plate.

The devices and methods described herein may be used, in someembodiments, to grow cells. For example, single cells may be grown ineach pot and/or droplet. Multiple cells may also be grown in each potand/or droplet in some cases. Cell growth may also arise, in some cases,from a single original cell or from two or more original cells. In somecases, multiple cells of different species may be co-encapsulated (e.g.,for directed coevolution). In some embodiments, droplets and/or pots maybe used to grow multicellular organisms (e.g. C. elegans). In someinstances, pots and/or droplets may include one or more suspensioncells. In some cases, pots and/or droplets may include one or moreadherent cells. In some cases, adherent cells are grown on beads withinthe pot and/or droplet. The devices and methods described herein may beused to determine the property of a droplet and/or its contents. In someembodiments, properties of droplets may be determined while they areconfined to pots. In some embodiments, extraneous substances (e.g.drugs) may be flowed through the channels and around the droplets duringtheir confinement. This may be accomplished by flowing the substances atflow rates low enough to prevent deformation of the cells. In otherembodiments, properties of droplets may be determined after they havebeen transferred to a secondary device (e.g., the well of a microtiterplate or a DNA microarray). As a non-limiting example, a microarrayscanner may be used to measure the fluorescence of a droplet thatresides in a pot. As another example, a camera can be used to record animage of the contents of pots. Properties of droplets that may bedetermined (in pots, secondary devices, or in some other location)include fluorescence, changes in phase within a droplet (e.g.nucleation, freezing), the level of gene expression, the amount of achemical secreted by a cell, the amount of a chemical taken up by acell, the number of positive reactions in an array of droplets, changesin morphology or shape or structure of encapsulated contents (e.g.cells, packing of colloidal particles, etc.), whether a cell is alive ordead, the motion of a cell, the motility of a cell, the reaction of acell to drug exposure, the phenotype of a cell (e.g., from an image of acell), the number of cells in a droplet, and the growth rate of cells,among others.

Systems and methods of the current invention may be used to monitor oneor more droplets as a function of time. As an example, a device maycomprise an array of pots each filled with one droplet. Each dropletremains in one location on the chip, in this case, and the time courseof its contents can be monitored, for example, by imaging the samplewith a camera. As another example, suspension cells can be immobilized,and their responses may be tracked over time. In some cases, thedroplets and/or their contents (e.g., one or more cells) may be kept inthe field of view and in the focal plane of an imaging device (e.g., acamera, microscope, etc.) while the droplets and/or cells remainconfined in pots. This may be important, for example, in the case ofcells that cannot be immobilized without showing changes in theirbehavior/metabolism. As another example, one may excite fluorescence inthe sample (e.g., with a laser, UV lamp, ACR lamp, LED, and/or halidelamp, among others) and monitor the intensity over time. Otherinstruments may be used to measure one or more properties of thecontents of one or more pots including, but not limited to, scanningdetectors such as PMTs, confocal microscopy, and spectroscopy amongothers. In some cases, many droplets can be monitored simultaneously,for example, by using a camera with a wide-angle view or by moving thedevice relative to the imaging instrument to sequentially image thedroplets in different wells. The ability to monitor droplets as afunction of time allows the user to calculate properties such as, forexample, reaction rates, growth rates, survival, and phenotype, amongothers. In one embodiment, differences in reaction rates among two ormore droplets may be used as the basis to sort those droplets. Inanother set of embodiments, the time evolution of fluorescence inindividual pots and/or droplets containing single cells can be used aspart of a digital PCR or real-time PCR analysis. In some cases,monitoring growth of cells in pots and/or droplets allows for fitness ortoxicity assays to be performed at the single cell level. In still othercases, single cells and their progeny remain isolated in pots and/ordroplets, and microcolonies deriving from single cells can be maintainedover time. Cell tracking can also be performed in cases where cellgrowth originates from multiple cells.

In some embodiments, droplets remain in pots during and/or after aperturbation. In one embodiment, droplets remain in their pots while thedevice is moved. For example, droplets may remain in their pots whilethe device is transported or shipped from one lab to another. In someembodiments, droplets may remain in pots while the device is moved fromone table to another. In some cases, the devices may be transported toand stored in a drawer or an incubator overnight or for several days.Droplets may also remain in their pots, for example, after the device isdropped on the floor. In some embodiments, droplets are maintained intheir pots as the device is heated. In some cases, droplets remain intheir pots as the device is heated to a temperature of at least about50° C. In other cases, droplets remain in their pots as the device isheated to a temperature of at least about 100° C. or at least about 200°C.

In some embodiments, the environment of the device may be controlled. Insome cases, the temperature of the device may be controlled, e.g., whenperforming PCR analysis. The device may be thermally cycled by, forexample, placing the device on a hot plate at the desired temperature.In some embodiments, the device may be substantially isothermal. Inother cases, heating may be achieved in a localized area by using, forexample, resistive heating.

In some cases, one or more of the walls of the device may comprise aselectively permeable material. For example, one or more walls of thedevice may comprise a water-permeable material. As another example, oneor more walls of the device may comprise a gas-permeable material,allowing for gas exchange across the surface. The selectively permeablematerial may be located between pots, in some embodiments. For example,the placement of permeable material between two or more pots may allowfor the exchange of media (e.g., a buffer, etc.) between pots. Theselectively permeable material may separate the contents of the pot froma controlled environment in some cases. In some embodiments, theselectively permeable material may separate the contents of the pot fromthe ambient atmosphere. The device may also be easily moved to anincubator. In some cases, the use of a selectively permeable materialmay lead to the unwanted evaporation of liquid (e.g., water) from thedevice. The evaporative effect may be minimized, in some cases, bysealing the device (e.g., with a glass slide) and saturating theselectively permeable material with liquid (e.g., water) to preventevaporation. In some embodiments, the evaporation of liquid may bedesirable. For example, the evaporation of liquid from the devicechannels may lead to changes in concentrations in the droplets. In somecases, the evaporation of the liquid from the device channel can becontrolled, leading to controlled concentration variation within thedroplets. This technique may be useful in applications such as, forexample, protein crystallization.

In one set of embodiments, the droplets may contain cells or otherentities, such as proteins, viruses, macromolecules, particles, etc.Cells may also constitute droplets in some embodiments. As used herein,a “cell” is given its ordinary meaning as used in biology. One or morecells and/or one or more cell types can be contained in a droplet.Cells, for example, can be suspended in a fluid such as, for example, anaqueous buffer solution or contained in a polymerosome. If apolymerosome is used, the shell surrounding the cell may be formed of amaterial capable of protecting the cell. The shell may help retain, forexample, moisture, and can be sized appropriately to maximize thelifetime of the cell within the polymerosome.

The cell may be any cell or cell type. For example, the cell may be abacterium or other single-cell organism, a plant cell, or an animalcell. The cell may be, in some cases an adherent cell and, in othercases, a suspension cell. If the cell is a single-cell organism, thenthe cell may be, for example, a protozoan, a trypanosome, an amoeba, ayeast cell, algae, etc. If the cell is an animal cell, the cell may be,for example, an invertebrate cell (e.g., a cell from a fruit fly), afish cell (e.g., a zebrafish cell), an amphibian cell (e.g., a frogcell), a reptile cell, a bird cell, or a mammalian cell such as aprimate cell, a bovine cell, a horse cell, a porcine cell, a goat cell,a dog cell, a cat cell, or a cell from a rodent such as a rat or amouse. If the cell is from a multicellular organism, the cell may befrom any part of the organism. For instance, if the cell is from ananimal, the cell may be a cardiac cell, a fibroblast, a keratinocyte, aheptaocyte, a chondracyte, a neural cell, a osteocyte, a muscle cell, ablood cell, an endothelial cell, an immune cell (e.g., a T-cell, aB-cell, a macrophage, a neutrophil, a basophil, a mast cell, aneosinophil), a stem cell, etc. In some cases, the cell may be agenetically engineered cell. In certain embodiments, the cell may be aChinese hamster ovarian (“CHO”) cell or a 3T3 cell. In some cases,multicellular organisms (e.g. C. elegans) may be used in the embodimentsdescribed herein.

As used herein, the term “average diameter” of a plurality or series ofdroplets is the arithmetic average of the average diameters of each ofthe droplets. Those of ordinary skill in the art will be able todetermine the average diameter (or other characteristic dimension) of aplurality or series of droplets, for example, using laser lightscattering, microscopic examination, or other known techniques. Theaverage diameter of a single droplet, in the case of a non-sphericaldroplet, is the diameter of a perfect sphere having the same volume asthe non-spherical droplet.

As used herein, two fluids are “immiscible,” or not miscible, with eachother when one is not soluble in the other to a level of at least 10% byweight at the temperature and under the conditions at which the multipleemulsion is produced. For instance, two fluids may be selected to beimmiscible within the time frame of a particular technique carried outin accordance with the invention.

The term “determining,” as used herein, generally refers to the analysisor measurement of a species, for example, quantitatively orqualitatively, and/or the detection of the presence or absence of thespecies. “Determining” may also refer to the analysis or measurement ofan interaction between two or more species, for example, quantitativelyor qualitatively, or by detecting the presence or absence of theinteraction. Examples of suitable techniques include, but are notlimited to, spectroscopy such as infrared, absorption, fluorescence,UV/visible, FTIR (“Fourier Transform Infrared Spectroscopy”), or Raman;gravimetric techniques; ellipsometry; piezoelectric measurements;immunoassays; electrochemical measurements; optical measurements such asoptical density measurements; circular dichroism; light scatteringmeasurements such as quasielectric light scattering; polarimetry;refractometry; or turbidity measurements.

The following examples are intended to illustrate certain embodiments ofthe present invention, but do not exemplify the full scope of theinvention.

EXAMPLE 1

This example describes a device used to arrange droplets inpredetermined locations. FIGS. 3 and 6 are optical micrographs ofarticles according to one embodiment of the invention. In this case, thepots comprise cylindrical chambers. The pots are arranged in a series ofstraight channels, with each pot in fluidic communication with exactlytwo constrictions.

The device in this example is fabricated by forming the features in PDMSusing a mold. Soft lithography was used to fabricate microfluidicchannels in polydimethylsiloxane (PDMS, Sylgard 184 Silicone Elastomer,Dow Corning, Midland, Mich., USA). The desired design was printed onto atransparency with features no smaller than 10 μm (CAD/Art Services,Inc., Bandon, Oreg.). SU8-2025 (Microchem, Newton, Mass., USA) wasspincoated onto a cleaned silicon wafer to a final thickness of 25 μmfollowing the protocol described by the manufacturer. Exposure to UVlight (200-250 mJ, OAI, San Jose, Calif.) crosslinked the exposedpattern, and the non-exposed photoresist is dissolved away usingpropylene glycol monomethyl ether acetate (PGMEA). Degassed PDMS with10% (w/w) crosslinking agent was then poured onto the SU8-mold. Afterheating at 65° C. for 1 hour, the structure was carefully peeled off themold, plasma-treated, and bonded to a 1×3″ glass slide (1.2 mmthickness). Holes connecting to the channels were formed using biopsypunches (Harris Uni-Core, 0.75 mm diameter). Polyethylene tubing (PE-20,VWR) was inserted into the holes. Before use, channels were treated withAquapel (The Tire Rack, South Bend, Ind.) followed by a flush with air.This treatment ensured that the oil carrier phase, and not the aqueousphase, wetted the surface.

During prolonged droplet storage, the absorbance of water by PDMS maycause droplets to shrink. To counter this problem, small glasscoverslips were placed directly above the storage areas of the device bysubmerging them in the liquid PDMS before curing. This served to reducethe volume of PDMS in direct contact with the storage channels.Additionally, water channel surrounding the entire storage area of theDropspots device was fabricated. The water in the channel saturated thesurrounding PDMS and prevented evaporation of water from the device. Inthis case, essentially constant humidity was maintained in the pots,thus preventing the droplets from shrinking when stored in the device.No observable shrinkage was observed, even when droplets were stored fordays.

In this embodiment, droplets were formed at rates of 1-10 kHz using anozzle (not shown) in the area to the left of the image. Droplets wereflowed into the channels and squeezed through the constrictions in thepresence of flow. When fluid flow was stopped, droplets stopped in thepots. In some cases, single cells were placed in the droplets by tuningthe density of the cell suspension such that 1 or 0 cells were placed ineach droplet.

In some cases, a bypass channel was incorporated into the device. Inthese cases, droplets can flow either into the array of pots or thebypass channel. This configuration allowed for the adjustment of theflow speed of the droplets as they entered the storage array. In thisexample, flow speed was adjusted by manually controlling the pressureapplied to the bypass channel inlet. In this way, the proportion ofdroplets entering the bypass channel (e.g., waste) was controlled. Thebypass channel, in some cases, also allows the user to easily stopfilling the storage array by unplugging the tubes. In providing anadditional outlet of low hydrodynamic resistance, the bypass channel mayserve to dampen the abrupt pressure change, and thus prevent the arrayof droplets from being disturbed.

Once the flow of droplets into the pots array is stopped, droplets weretrapped in the pots. Not wishing to be bound by any theory, once thedroplets were trapped in the pots, they may have achieved their minimalenergy shape, a sphere. Any deviations from a spherical shape may createan increase in surface area, and thus free energy. Droplets are therebyprevented from entering the constrictions between different pots. FIG. 4is an optical micrograph of droplets trapped in pots as described inthis example. In this set of experiments, the ratio of the diameters ofthe pots to constrictions had to be greater than ˜2:1. In cases wherethe constrictions were larger than this, droplets were able to squeezethrough and did not remain in single pots over the course of theexperiment.

The device and method of droplet storage described in this example wasproven to be stable and robust. The device was moved, heated in anincubator and on a hot plate, and imaged without affecting the positionof droplets. The large surface area-to-volume of the droplets, as wellas the permeability of PDMS to gases, made this device especiallyamenable to cell culture under specific environmental conditions. Thedevice described in this example also easily interfaced with standardequipment in biological facilities: the device can be adapted to fitinto a microarray scanner or onto the stage of an inverted microscope.Microscopes equipped with an automated stage enable thousands ofdroplets to be imaged over time. After the experiment, droplets wereeasily recovered from the storage device by connecting tubing andinjecting oil into the device.

EXAMPLE 2

To demonstrate the capability to perform time-lapse measurements using adevice according to one embodiment of the invention, growth rates in apopulation of single yeast cells were monitored. Yeast cell cultures (S.cerevisiae, MATa s288c) were inoculated from a single colony and grownfor 6 hours in YPD media at 30° C. A 10 μL aliquot of this stock culturewas diluted in 10 mL of YPD and cultured overnight to a density ofOD600˜0.02-0.06 or 0.2-0.6 (1 mm path length, NanoDrop ND-1000,Wilmington, Del.). Cells are washed twice by centrifugation, resuspendedin fresh YPD, and then encapsulated.

Yeast cells were encapsulated in droplets of water-in-fluorocarbonemulsion, and immobilized in pots on a device. To achieve theencapsulation, the cell suspension was loaded into 1 mL plastic syringes(BD) fitted with a 27¾ gauge needle (Luer-lock, 27¾ gauge). PE-20 tubingwas inserted onto the needle, and the tubing was plugged into theexcised hole on the PDMS device. To encapsulate single cells indroplets, cell suspensions were flowed together with oil (Fluorinert(FC-40 Sigma) containing 1.8% fluorinated surfactant (Holtze et al, inpreparation) using flow-focusing geometry 14 to generate monodispersedroplets of a water-in-oil emulsion. Syringe pumps (New Era Pump SystemsInc., Farmingdale, N.Y.) were used to control flow rates: 100 μl/hr forthe aqueous phase and 300 μl/hr for the oil phase results in droplets of˜20 μm diameter.

Once the cells were encapsulated and the droplets were arranged in pots,the device containing the cells was incubated overnight. An array of 160pots was monitored over 15 hours, with images acquired every 10 minutes.FIG. 5 illustrates a typical encapsulation experiment. In this example,151 pots were filled with droplets, and 71 of those contained one ormore cells. The presence of multiple cells in a droplet did not pose aproblem, as individual droplets were tracked from time zero, and thusdroplets that contained single cells were selected for analysis. Thetotal number of cells increased from 158 to 444. In seven droplets nocell division was observed over the total 15-hour time period. FIG. 7Ais an optical micrograph showing the increase in number of cells in oneexperiment. FIG. 7B includes a plot of the cell growth in pots B7, B8,and C7 during the experiment shown in FIG. 7A.

Plotting the total number of cells as function of time yielded typicalsigmoidal growth curves observed for bulk cell culture. The initialgeneration time was lower than typical values for bulk growth. Notwishing to be bound by any theory, this may have been due to the high“cell density” in the droplets. The volume per cell of a single cell ina droplet of 40 micron diameter is equivalent to 108 cells per mL, whichis near the density of a saturated culture of yeast cells. The observeddelay in initial division may also have been due to the lag phase.

The generation time for encapsulated cells as function of initiallyencapsulated cell number was also examined. The doubling time increasedwith increasing cell number. Although mean values of doubling timeclearly followed the trend mentioned above, the doubling time variedsignificantly among cells in droplets, even when identical numbers ofcells are encapsulated. This demonstrates the heterogeneity of cellbehavior at the single cell level, one major factor asynchronization ofthe cell cycle in individual cells.

During timecourse experiments, it was observed that droplets shrank involume. This may have been due to absorbance of water by the PDMS. Toprevent droplets from shrinking, the PDMS could be, in some cases,saturated with media. This was accomplished by filling the water channelreservoir. A cover glass was placed on top of the channels to furtherprevent droplet shrinkage. When this scheme was adopted, the volumes ofdroplets containing cell media (YPD) remained constant. In an array ofdroplets containing cells, however, the volume of empty dropletsincreased, whereas droplets containing cells shrank as a function oftime. Interestingly, the final volume of the droplets decreases with thenumber of initially encapsulated cells.

Not wishing to be bound by any theory, the following explanation mayexplain the behavior: cells inside droplets may have consumed moremolecules than they excreted, and thus, the osmolarity of the mediainside the droplet may have been lowered. This may have led to anosmotic pressure difference which may have forced water from dropletscontaining cells into empty droplets. This could be a very interestingtool to easily observe metabolic rates at the single cell level.

EXAMPLE 3

Droplets are helpful in studying populations of single cells andcompounds they secrete as high concentrations of molecules are rapidlyattained in such small droplet volumes. Secreted enzymes have been usedas reporters for gene expression, both for high-throughput screening aswell as for fundamental studies of enzyme kinetics and gene expression.To demonstrate the utility of droplets as microvessels for secretedenzyme detection at the single cell level, single cells wereencapsulated producing beta-galactosidase, and monitored production offluorescein (FIG. 8A) as the enzyme cleaved its fluorogenic substrate,fluorescein-di-beta-D-galactopyranoside (FDG). FIG. 8A includes aBrightfield image and color map gradient of a fluorescence image at time¼ 45 min. For cells in the picoliter-volume droplets of a Dropspotsarray, the signal was detected after minutes. Slope analysis in thelinear regime of the enzyme progress curves showed that reaction ratesdepend on the number of cells per droplet, but vary even for dropletscontaining the same number of cells, reflecting heterogeneity in geneexpression in a population of isogenic cells (FIGS. 8B-C). FIG. 8Bincludes plots for 9 individual representative droplets containing 1, 2,and 3 cells. Reaction rates for 265 droplets containing cells werequantified by slope analysis in the linear regime, and displayed as afunction of number of cells per droplet in the histogram shown in FIG.8C. A total of over 2000 droplets were analyzed, but data is displayedonly for droplets that contained cells. The scale bar in FIG. 8Crepresents 40 mm. Previous studies of enzyme kinetics at the single celllevel have been limited by their dynamic range, as detectable levels ofenzyme must first accumulate in the encapsulation volume before analysiscan begin.

EXAMPLE 4

Lipid vesicles provide a model system to study membranes and lipidbilayers. To avoid the effects of substrate interactions, free-floatinglipid vesicles are desirable for use in studies of lateral membraneorganization. They are also well-suited for use as biocompatible vesselsfor encapsulation and targeted drug delivery. In order to stabilizevesicles for studying their physical properties such as lateralorganization by confocal microscopy, or permeability, extraneouscompounds that may alter lipid bilayer properties (e.g., sugars) areadded to the buffer to gravitationally stabilize the vesicles. TheDropspots device may be used to trap lipid vesicles without the additionof exogeneous substances. Permeability can be studied by monitoring bymicroscopy the volume changes in vesicles in response to osmoticchanges. An immobilized array of vesicles would also enable the study ofdynamic behavior of encapsulated contents, where the lipid vesicleserves as an in vitro compartment.

While several embodiments of the present invention have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, kit, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,kits, and/or methods, if such features, systems, articles, materials,kits, and/or methods are not mutually inconsistent, is included withinthe scope of the present invention.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

What is claimed is:
 1. A method of arranging cells in an array, themethod comprising: flowing cells through a plurality of storage channelsarranged in series, wherein each storage channel comprises one or morepots each comprising a constriction that is smaller than an averagecross-sectional dimension of a single cell, wherein each pot has no morethan two constrictions and is in direct fluid communication with no morethan two other pots; trapping a single cell in each of the one or morepots in a first of the plurality of storage channels; and flowing cellsaround the first of the plurality of storage channels through one ormore bypass channels.
 2. The method of claim 1, wherein the volumes ofthe one or more pots are about 10 nanoliters.
 3. The method of claim 1,further comprising separating the contents of the one or more pots froma controlled environment by a gas-permeable membrane.
 4. The method ofclaim 1, further comprising separating the contents of the one or morepots from ambient atmosphere by a gas-permeable membrane.
 5. The methodof claim 1, further comprising performing a reaction on the trappedsingle cell.
 6. The method of claim 5, wherein the one or more pots areassociated with a thermal cycler, wherein the reaction comprises PCR. 7.The method of claim 6, wherein the PCR is digital PCR.
 8. The method ofclaim 6, wherein the PCR is real-time PCR.
 9. The method of claim 1,wherein each pot is connected to exactly two constrictions.
 10. Themethod of claim 1, wherein the plurality of storage channels are part ofa device comprising at least 100 pots.
 11. The method of claim 1,wherein the single cell is a bacterium cell.
 12. The method of claim 1,wherein the single cell is an immune cell.
 13. The method of claim 1,wherein the single cell is a T cell, B cell, a macrophage, a neutrophil,a basophil, a mast cell, or an eosinophil.
 14. The method of claim 1,wherein the single cell is an engineered cell.